Paraquat resistance gene and a vascular tissue - and trichome-specific promoter

ABSTRACT

A paraquat resistance gene and a vascular tissue- and trichome-specific promoter are provided. The paraquat resistance gene and the vascular tissue- and trichome-specific promoter are isolated by identifying and analyzing genes of  Arabidopsis thaliana.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a paraquat resistance gene impartingparaquat resistance and a vascular tissue- and trichome-specificpromoter.

2. Background Art

Plants are exposed to various environmental stresses on a regular basisincluding high and low temperatures, drought, high light intensity,salinity, air pollutant gases, pathogenic microbes and the like.Therefore, if useful plants that can grow sufficiently even under suchtypes of environmental stresses such as, for example, crops, can bedeveloped, food production will become possible even in regions in whichcrops and the like can not currently grow due to environmental stresses,and the possibility of being prepared for a grave food crisis that isforecast in the future will be increased. Consequently, the productionof plants that have improved resistance to such kinds of environmentalstresses is underway on a global basis. For example, plants have beenproduced that were imparted with chilling resistance (Nature, 356,710-703, 1992; Plant Physiol., 105, 601-605, 1994), drought resistance(Plant Physiol., 107, 125-130, 1995; Nature, 379, 683-684, 1996; NatureBiotech., 17, 287-291, 1999), salt resistance (Science, 259, 508-510,1993; Biotechnology, 14, 177-180, 1996; Plant J., 12, 133-142, 1997),air pollutants resistance (Plant Cell Physiol., 34, 129-135, 1993;Biotechnology, 12, 165-168, 1994), disease resistance (Kagaku toSeibutsu (Chemistry and Organisms), 37, 295-305, 385-392, 1999) and thelike by genetic recombination techniques. Further, some plants that havebeen imparted with resistance to agricultural chemicals by geneticrecombination techniques are in practical use (Nature, 317, 741-744,1985; Proc. Natl. Acad. Sci. USA, 85, 391-395, 1988; EMBO J., 6,2513-2518, 1987; EMBO J., 7, 1241-1248, 1988).

These environmental stresses are closely related with in vivo generationof active oxygen species (superoxide radical (O₂ ⁻), hydrogen peroxide(H₂O₂), hydroxy radical (OH⁻)). Active oxygen species are generated byrespiration, photosynthesis, environmental stresses and the like, andimpart fatal damage to cells by excessive oxidation of proteins, nucleicacids, membrane structure or the like. It has also been reported that anactive oxygen-resistant plant produced by genetic recombinationtechniques showed improved resistance to the aforementionedenvironmental stresses (Plant Physiol., 111, 1177-1181, 1996; FEBSLetters, 428,47-51, 1998).

To produce an active oxygen-resistant plant, a method is principallyemployed in which a gene of enzyme scavenging active oxygen species(superoxide dismutase, ascorbate peroxidase, catalase and glutathionereductase and the like) is introduced into the plant.

Paraquat is a non-selective and potent herbicide that can kill allplants by continuously generating active oxygens in the photosystems.Paraquat resistance can thus be used as an indicator of the resistanceto active oxygens, and analysis concerning the mechanism of paraquatresistance in plants has been conducted (Pestic. Biochem. Physiol., 26,22-28, 1986; Theor. Appl. Genet. 75, 850-856, 1988; and Plant Physiol.,91, 1174-1178, 1989).

Meanwhile, an apoptosis suppressor gene (JP Patent Publications (Kokai)No. 10-309142; No. 2000-23583; and No. 2002-300822), a gene encoding aprotein homologous to aldose reductase (JP Patent Publication (Kohyo)No. 2001-523466) and a gene encoding an iron-binding protein (ferritin)(JP Patent Publication (Kohyo) No. 2001-519671) have been disclosed asgenes that can impart paraquat resistance. Further, in JP PatentPublications (Kokai) No. 2002-281979 and No. 2001-95585, peroxidasederived from paraquat resistant callus is disclosed as a gene capable ofimparting resistance to paraquat.

It had been believed that if a paraquat resistance gene that can impartstrong resistance to paraquat could be isolated, it would be useful inthe development of plants with high resistance to active oxygensgenerated under various kinds of environmental stress conditions (highand low temperatures, drought, high light intensity, salinity, airpollutant gases, pathogenic microbes and the like). However, recently ithas been revealed that active oxygens fulfill an important role as amolecule regulating the growth and stress response of a plant.Therefore, to avoid influencing important characteristics such as cropyield, it is important to increase the resistance of a plant to stressessuch as paraquat without affecting the growth and physiological controlmechanisms of a plant dependent on active oxygens.

The vascular tissue is a fascicular tissue system that differentiatesthrough each organ of pteridophytes and spermatophytes, such as thestem, leaf and root. Xylem and phloem are the components of the vasculartissue, and they function as pipes to transport water and internalsubstances throughout the plant. Further, the vascular cambium, whichincludes the interfascicular cambium and the intrafascicular cambium, isfound in the vascular tissue. The vascular cambium is a site of cellproliferation, and is thus an extremely important site for the growth ofa plant. Thus, the vascular tissue is a location involved intransporting water and internal substances as well as cell proliferationin a plant. Accordingly, if a gene involved in transporting water orinternal substances or in cell proliferation can be introduced into aplant and expressed specifically in the vascular tissue, it will bepossible to regulate the transport of water or internal substances orcell proliferation in the plant.

In addition, from the viewpoint of plant diseases, the vascular tissueis a site where a wilt disease fungus infecting plants of the familySolanaceae proliferates and transfers. When a plant virus infects aplant, the plant virus migrates a long distance from one leaf to anabove leaf, and therefore the vascular tissue is also a migration sitethat leads to systemic infection of a plant. Accordingly, if a geneinvolved in proliferation or migration of a fungus or plant virus can beintroduced into a plant and expressed specifically in the vasculartissue, the plant can be protected from the fungus or plant virus.

A trichome is a floccose outgrowth found on the surface of a leaf, stem,sepal and the like of a plant body. A trichome is involved in secretionand excretion from the surface of a plant body. For example, it isreported that when a plant is exposed to heavy metal (cadmium) stress,the number of trichomes on the surface of leaves increases and crystalscontaining cadmium or calcium adhere to the surface of the leaves, inother words, that cadmium is excreted by a trichome (Planta, 213 (1),45-50, 2001, May). A trichome is also the site of first contact for afilamentous fungus, bacterium, insect or the like invading a plant.Further, as a defense against diseases and insect damages, for example,a fluid having antimicrobial activity and a feeding deterrent effect issecreted from a glandular hair or glandular trichome of rugosa rose ofthe family Rosaceae, one type of trichome. Therefore, if a gene involvedin the excretion of a heavy metal or the like, or a gene involved in thesecretion of a fluid having antimicrobial activity or a feedingdeterrent effect can be introduced into a plant and expressedspecifically in a trichome, a heavy metal can be efficiently excretedfrom the plant or the plant can be effectively protected against afilamentous fungus, bacterium or insect invading the plant.

As described above, it is desirable that specific gene expression beperformed in a vascular tissue or trichome. As a method for performingspecific gene expression, a method involving the use of a promoterexhibiting specific promoter activity in a vascular tissue or trichomecan be considered. However, a promoter exhibiting promoter activityspecifically in both a vascular tissue and a trichome has not beenidentified at present.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide, for example, aparaquat resistance gene and a vascular tissue- and trichome-specificpromoter by identifying and analyzing genes of Arabidopsis thaliana.

The present invention accomplishes the aforementioned object byproviding the following:

(1) a gene encoding a protein of the following (a) or (b):

-   (a) a protein consisting of the amino acid sequence represented by    SEQ ID NO: 2;-   (b) a protein consisting of an amino acid sequence having a    substitution, deletion or addition of one or a plurality of amino    acids relative to the amino acid sequence represented by SEQ ID NO:    2 and imparting paraquat resistance;

(2) a protein imparting paraquat resistance encoded by the gene asrecited in the above (1);

(3) a recombinant vector comprising the gene as recited in the above(1);

(4) the recombinant vector according to (3), wherein the recombinantvector further comprises a foreign gene or a foreign DNA fragment;

(5) a transformant having the recombinant vector as recited in the above(3) or (4);

(6) a plant body having the recombinant vector as recited in the above(3) or (4) and having paraquat resistance;

(7) a method for screening for a transgenic plant, comprisingintroducing a recombinant vector as recited in the above (4) into aplant and screening for a transgenic plant on the basis of paraquatresistance as an indicator;

(8) a vascular tissue- and trichome-specific promoter comprising DNA ofthe following (a), (b) or (c):

-   (a) DNA consisting of the nucleotide sequence represented by SEQ ID    NO: 3;-   (b) DNA consisting of a nucleotide sequence having a substitution,    deletion or addition of one or a plurality of nucleotides relative    to the nucleotide sequence represented by SEQ ID NO: 3 and    functioning as a vascular tissue- and trichome-specific promoter;    and-   (c) DNA hybridizing under stringent conditions to DNA consisting of    the nucleotide sequence represented by SEQ ID NO: 3 and functioning    as a vascular tissue- and trichome-specific promoter;

(9) a recombinant vector comprising the vascular tissue- andtrichome-specific promoter as recited in the above (8);

(10) the recombinant vector according to (9), wherein the recombinantvector comprises a foreign gene or a foreign DNA fragment downstream ofthe vascular tissue- and trichome-specific promoter;

(11) the recombinant vector according to (10), wherein the foreign geneis the gene as recited in the above (1); and

(12) a transgenic plant having the recombinant vector as recited in anyone of the above (9) to (11).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph of electrophoresis of cDNA derived from an AtMVRgene transformant;

FIG. 2A is a schematic diagram showing the location of an AtMVR genetransformant and a non-transformant in FIGS. 2B and 2C. FIG. 2B is aphotograph showing the growth of an AtMVR gene transformant and anon-transformant in a ½ MS culture medium without paraquat. FIG. 2C is aphotograph showing the growth of an AtMVR gene transformant and anon-transformant in a ½ MS culture medium with paraquat;

FIG. 3 is a photomicrograph of an entire transformant containing the GUSgene and an AtMVR promoter histochemically colored with GUS;

FIG. 4 is a photomicrograph of a leaf of a transformant containing theGUS gene and an AtMVR promoter histochemically colored with GUS; and

FIG. 5 is a photomicrograph of a root of a transformant containing theGUS gene and an AtMVR promoter histochemically colored with GUS.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention will be described in detail below.

The gene according to the present invention is a gene encoding theprotein of the following (a) or (b):

-   (a) a protein consisting of the amino acid sequence represented by    SEQ ID NO: 2; and-   (b) a protein consisting of an amino acid sequence having a    substitution, deletion or addition of one or a plurality of amino    acids relative to the amino acid sequence represented by SEQ ID NO:    2 and imparting paraquat resistance.

The gene encoding the protein described in the above (a) is a gene(hereafter, referred to as “AtMVR gene”) encoding a protein impartingparaquat resistance which consists of the amino acid sequencerepresented by SEQ ID NO: 2.

The present inventors performed a search on databases having the entirenucleotide sequence of Arabidopsis thaliana (for example, GenBank, EMBL,DDBJ, tair: The Arabidopsis Information Resource) based on thenucleotide sequence of the AtMVR gene and found that there are 13 geneshomologous to the AtMVR gene (AtMVR 3-1 to AtMVR 3-13) present on theArabidopsis thaliana genome. The nucleotide sequence of each of theseAtMVR homologous genes and the putative amino acid sequence encoded bythe relevant AtMVR homologous gene are represented by the SEQ ID NOs.listed in Table 1 below. Table 1 also lists the results of homologyanalysis between the AtMVR gene and each AtMVR homologous gene. Thehomology analysis was conducted using BLAST P at the amino acid level.Amino acids may be classified based on the chemical properties of theirside chains. In the BLOSUM62 amino acid substitution matrix (Proc. Natl.Acad. Sci., 89, 10915-10919, 1992), amino acids are classified into: anamino acid with a mercapto group (C); hydrophilic amino acids that havelow molecular weight (S, T. P, A, G); acidic amino acids (N, D, E, Q);basic amino acids (H, R, K); hydrophobic amino acids that have lowmolecular weights (M, I, L, V); and aromatic amino acids (F, Y, W). InTable 1, the term “Identities” refers to 100% correspondence in terms ofamino acids and the term “Positives” refers to the numerical value whenamino acids having a positive score in the BLOSUM62 amino acidsubstitution matrix are added to those having 100% correspondence (seeBioinfomatics (in Japanese), Eds. Okazaki Y. & Bono H. (published byMedical Science International)). TABLE 1 Name of AtMVR homologous geneNucleotide sequence Amino acid sequence Identities (%) Positives (%)AtMVR3-1 SEQ ID NO: 4 SEQ ID NO: 5 57 70 AtMVR3-2 SEQ ID NO: 6 SEQ IDNO: 7 55 69 AtMVR3-3 SEQ ID NO: 8 SEQ ID NO: 9 39 56 AtMVR3-4 SEQ ID NO:10 SEQ ID NO: 11 39 55 AtMVR3-5 SEQ ID NO: 12 SEQ ID NO: 13 37 54AtMVR3-6 SEQ ID NO: 14 SEQ ID NO: 15 36 52 AtMVR3-7 SEQ ID NO: 16 SEQ IDNO: 17 34 52 AtMVR3-8 SEQ ID NO: 18 SEQ ID NO: 19 34 51 AtMVR3-9 SEQ IDNO: 20 SEQ ID NO: 21 37 58 AtMVR3-10 SEQ ID NO: 22 SEQ ID NO: 23 25 44AtMVR3-11 SEQ ID NO: 24 SEQ ID NO: 25  33*  51* AtMVR3-12 SEQ ID NO: 26SEQ ID NO: 27 25 41 AtMVR3-13 SEQ ID NO: 28 SEQ ID NO: 29 22 41*The comparison with AtMVR3-11 shows the homology result for comparisonwith a partial sequence of AtMVR3-11.

As shown in Table 1, the homology of AtMVR with the 13 AtMVR homologousgenes ranged from 22 to 57% for Identities and from 41 to 70% forPositives. These AtMVR homologous genes are considered to impartparaquat resistance in the same manner as the AtMVR gene.

Further, the AtMVR gene has homology to a senescence-associated protein,DSA 5 (GenBank accession number AF082030) (Plant Molecular Biology 40,237-248, 1999). The result of homology analysis using BLAST X showed theprotein encoded by the AtMVR gene has identity of 89% at the amino acidlevel to the protein encoded by DSA 5. It is reported that DSA 5 is agene that expresses upon aging of the petal of lily (Hemerocallis hybridcultivar) (Plant Molecular Biology 40, 237-248, 1999). However, sincethe protein encoded by DSA 5 has no homology with any known protein, itis unclear which functions the protein has. Accordingly, the AtMVR geneis a novel gene imparting paraquat resistance.

As used herein, the term “paraquat resistance” refers to havingresistance to paraquat. More specifically, the term “paraquat-resistantplant” refers to a plant requiring a larger quantity of paraquat than anon-resistant plant in order to obtain a given effect from paraquat.Paraquat is a non-selective and potent herbicide that kills all plantsby continuously generating active oxygens in the photochemical system.It is possible to confirm whether the AtMVR gene is a paraquatresistance gene imparting paraquat resistance by examining whether atransformant into which the gene was introduced can grow in the presenceof paraquat.

The gene encoding the protein described in the above (b) is a geneencoding a protein consisting of an amino acid sequence having asubstitution, deletion or addition of one or a plurality of amino acids(for example, 1 to 10, or 1 to 5) relative to the amino acid sequencerepresented by SEQ ID NO: 2 and imparting paraquat resistance.

Once the nucleotide sequence of the gene according to the presentinvention has been determined, it is then possible to obtain the geneaccording to the present invention by chemical synthesis, or bypolymerase chain reaction (hereafter, referred to as “PCR”) employing asa template a clone that has been cloned, or by performing hybridizationemploying a DNA fragment having the nucleotide sequence as a probe.Further, it is possible to synthesize a mutant of the gene according tothe present invention having equivalent functions as those prior tomutation by a technique such as site-directed mutagenesis.

Examples of the method for introducing a mutation into the geneaccording to the present invention include a known method such as theKunkel method or the gapped duplex method or a method in accordance withsuch methods. For example, introduction of a mutation can be performedusing a kit for introducing a mutation (for example, Mutant-K(manufactured by TAKARA, Inc.), or Mutant-G (manufactured by TAKARA,Inc.)) utilizing site-directed mutagenesis or using LA PCR in vitroMutagenesis series kit manufactured by TAKARA, Inc.

A protein imparting paraquat resistance according to the presentinvention is the protein encoded by the gene according to the presentinvention. For example, the gene according to the present invention isintegrated into a vector derived from Escherichia coli or the like, andE. coli is then transformed with the obtained recombinant vector.Thereafter, the protein according to the present invention can beobtained by extracting the protein synthesized within E. coli.

Further, a recombinant vector according to the present invention is arecombinant vector comprising the gene according to the presentinvention. The recombinant vector according to the present invention canbe obtained by inserting the gene according to the present inventioninto an appropriate vector. A vector used for inserting the geneaccording to the present invention is not particularly limited as longas it is capable of replication within a host, and examples thereofinclude a plasmid, a shuttle vector, and a helper plasmid. In addition,when the vector itself is not capable of replication, a DNA fragmentthat is capable of replication by a method such as insertion into thechromosome of a host may be used.

Examples of plasmid DNA include a plasmid derived from E. coli (pBI221and the like, for example, pET system such as pET30b, pBR system such aspBR322 and pBR325, pUC system such as pUC118, pUC119, pUC18 and pUC19,pBluescript, and pBI221), a plasmid derived from Bacillus subtilis (forexample, pUB110 and pTP5), a binary plasmid derived from Agrobacteriumtumefaciens (for example, pBI system derived from pBIN19, pBI101, orpBI121), a plasmid derived from yeast (for example, YEp system such asYEp13, or YCp system such as YCp50) or the like. Examples of phage DNAinclude λ phage (Charon 4A, Charon 21A, EMBL3, EMBL4, λgt10, λgt11, λZAPand the like). Further, an animal virus vector such as retrovirus orvaccinia virus, a plant virus vector such as cauliflower mosaic virus,or an insect virus vector such as baculovirus can also be used.

To insert the gene according to the present invention into a vector, amethod may be used in which cDNA of the gene according to the presentinvention is first cleaved using an appropriate restriction enzyme andthen inserted into a restriction enzyme site or multicloning site of anappropriate vector DNA and ligated into the vector. Further, a methodmay be used in which a homologous region is respectively provided in onepart of a vector and cDNA of the gene according to the presentinvention, and the vector and the cDNA are connected by an in vitromethod using PCR or the like or an in vivo method using yeast or thelike.

A recombinant vector according to the present invention can also includea foreign gene or a foreign DNA fragment in addition to the geneaccording to the present invention. A method for inserting a foreigngene or a foreign DNA fragment into a vector is the same as the methodfor inserting a DNA fragment according to the present invention into avector. Any gene or DNA fragment may be used as a foreign gene or aforeign DNA fragment. Thus, the gene according to the present inventioncan be used as a selective marker gene to indicate paraquat resistance,for example, as with an antibiotic resistance gene for kanamycin orhygromycin or the like.

A transformant according to the present invention is a transformanthaving the recombinant vector according to the present invention. Thetransformant according to the present invention can be obtained byintroducing the recombinant vector according to the present inventioninto a host. A host is not particularly limited as long as it is capableof expressing the gene according to the present invention, however aplant is preferred. When the host is a plant, it is possible to obtain atransgenic plant in the manner described below.

A “plant” to be transformed in the present invention may be any of: awhole plant, a plant organ (for example, leaf, petal, stem, root, orseed), plant tissue (for example, epidermis, phloem, parenchyma, orxylem) or a plant culture cell. Examples of the plant that can be usedin the transformation include, but are not limited to, a plant belongingto the family Poaceae, Brassicaceae, Solanaceae, or Leguminosae (seebelow).

-   -   Poaceae: Oryza sativa, Zea mays    -   Brassicaceae: Arabidopsis thaliana    -   Solanaceae: Nicotiana tabacum    -   Leguminosae: Glycine max

The recombinant vector according to the present invention can beintroduced into a plant by a conventional transformation method such as,for example, the electroporation method, Agrobacterium method, particlegun method, or PEG method.

For example, when using the electroporation method, the recombinantvector according to the present invention is introduced into a host byconducting the treatment using an electroporation apparatus equippedwith a pulse controller under conditions of a voltage of 500 to 1600 V,at 25 to 1000 μF, for 20 to 30 msec.

When using the particle gun method, the whole plant, a plant organ orplant tissue itself may be used without any treatment, a section thereofmay be prepared and then used, or protoplast may be prepared and used.The prepared sample can then be treated using a gene transfer device(for example, PDS-1000/He manufactured by Bio-Rad Inc.). Although thetreatment conditions may vary depending on the plant or sample used, thetreatment is normally conducted at a pressure of approximately 450 to2000 psi and a distance of approximately 3 to 12 cm.

A method using the Ti plasmid or Ri plasmid of Agrobacterium takesadvantage of a characteristic whereby, when a bacterium belonging to thegenus Agrobacterium infects a plant, one part of plasmid DNA possessedby the bacterium is transferred into the genome of the plant. Thismethod can thus be used to introduce the gene according to the presentinvention into a plant host. Among the bacteria belonging to the genusAgrobacterium, when Agrobacterium tumefaciens infects a plant, it causesthe formation of a tumor that is referred to as “crown gall.” Further,when Agrobacterium rhizogenes infects a plant, it incites generation ofa capillary root. These are caused by a region referred to as a “T-DNA(Transferred DNA) region” of a Ti plasmid or Ri plasmid transferringinto a plant at the time of infection to be integrated into the genomeof the plant. Accordingly, the DNA to be integrated into a plant genomeis first inserted into the T-DNA region of a Ti plasmid or Ri plasmid,and then the DNA can be integrated into the plant genome by infectingthe plant host with a bacterium of the genus Agrobacterium.

Examples of the method for transforming a bacterium of the genusAgrobacterium into a plant host include the above describedelectroporation method, patent gun method and PEG method, as well as anin planta method. Examples of the in planta method include a directAgrobacterium inoculation method and an infiltration method.

Tumor tissue or shoot, capillary root or the like obtained as the resultof the transformation can be used without any treatment for cellculture, tissue culture or organ culture. Alternatively, it can beregenerated in a plant body by administration of a plant hormone (auxin,cytokinin, gibberellin, abscisic acid, ethylene, brassinolide or thelike) of an appropriate concentration using a conventional plant tissueculture method.

The gene according to the present invention may also be introduced intoa plant by utilizing a plant virus as a vector. Examples of the plantvirus that can be used include cauliflower mosaic virus. First, theviral genome is inserted into a vector derived from E. coli or the liketo produce a recombinant, and then the gene according to the presentinvention is inserted into the viral genome. The viral genome modifiedin this manner is subsequently cleaved from the recombinant using arestriction enzyme, and the gene according to the present invention canthen be introduced into a plant host by inoculating the viral genomeinto the plant host.

In addition to introduction into a plant host as described above, therecombinant vector according to the present invention may also beintroduced into bacteria belonging to the genus Escherichia such as E.coli, the genus Bacillus such as Bacillus subtilis, or the genusPseudomonas such as Pseudomonas putida, as well as yeast such asSaccharomyces cerevisiae and Schizosaccharomyces pombe, animal cellssuch as COS cell or CHO cell, and insect cells such as Sf9. When using abacterium such as E. coli or yeast or the like as a host, it ispreferable that the recombinant vector according to the presentinvention is capable of autonomous replication in the bacterium and thatit is comprised of a promoter, a ribosome binding sequence, atranscription termination sequence and the gene according to the presentinvention. It may also comprise a gene regulating the promoter.

The method for introducing the recombinant vector according to thepresent invention into a bacterium is not particularly limited as longas it is a method that can introduce DNA into a bacterium, and forexample a method using calcium ion or the electroporation method may bementioned.

The method for introducing the recombinant vector according to thepresent invention into yeast is not particularly limited as long as itis a method that can introduce DNA into yeast, and for example theelectroporation method, spheroplast method and lithium acetate methodmay be mentioned.

When using an animal cell as a host, monkey cell COS-7, Vero, Chinesehamster ovary cell (CHO cell), mouse L-cells or the like can be used.The method for introducing the recombinant vector according to thepresent invention into an animal cell is not particularly limited aslong as it is a method that can introduce DNA into an animal cell, andfor example the electroporation method, calcium phosphate method andlipofection method may be mentioned.

When using an insect cell as a host, an Sf9 cell or the like can beused. The method for introducing the recombinant vector according to thepresent invention into an insect cell is not particularly limited aslong as it can introduce DNA into an insect cell, and for example thecalcium phosphate method, lipofection method and electroporation methodmay be mentioned.

It is possible to confirm whether or not the gene according to thepresent invention has been integrated into a host by using the PCRmethod, Southern hybridization method, Northern hybridization method orthe like. For example, PCR can be conducted after preparing DNA from thetransformant and designing a DNA-specific primer. Next, theamplification product is subjected to agarose gel electrophoresis,polyacrylamide gel electrophoresis or capillary electrophoresis or thelike, and the product thereof is then stained with ethidium bromide,SYBR Green solution or the like. Thereafter, whether or nottransformation has occurred can be confirmed by the detection of theamplification product as a single band. It is also possible to detectthe amplification product after conducting PCR using a primer that hasbeen labeled previously with a fluorescent dye or the like. In addition,a method may be employed in which the amplification product is bound toa solid phase of a microplate or the like to enable confirmation of theamplification product by fluorescence or enzyme reaction or the like.

A plant body according to the present invention is one having arecombinant vector comprising the gene according to the presentinvention and having paraquat resistance. As used herein, the term“plant body” refers to a whole plant transformed with a recombinantvector comprising the gene according to the present invention. The plantbody according to the present invention can be obtained by introducingthe above recombinant vector into a plant cell or the like andregenerating a transgenic plant body from the obtained transgenic plantcell. As a regeneration method, a method may be employed in whichtransformed cells in a callus form are transferred to a culture mediumin which the type and concentration of hormones have been modified andallowed to culture, and an adventitious embryo is allowed to form toobtain a complete plant body. Examples of the culture medium to be usedinclude LS medium and MS medium. Introduction of a recombinant vectorinto a plant cell or the like can be performed by a method similar tothe method described above.

In the plant body according to the present invention, a proteinimparting paraquat resistance that is encoded by the gene according tothe present invention is overexpressed throughout the whole plant body.Thus, the plant body according to the present invention can haveresistance to paraquat.

A method of screening for transgenic plants according to the presentinvention is a method in which the recombinant vector according to thepresent invention is introduced into plants and paraquat resistance isused as an indicator to screen for transgenic plants. Transformation canbe verified by employing the gene according to the present invention asa selective marker gene to indicate paraquat resistance. Examples of thescreening method include a method in which plants transformed by therecombinant vector according to the present invention are grown in aparaquat-containing medium and the screening is carried out based onvariations in the life and death as well as growth of the plants. Theconcentration of paraquat used for the screening may vary depending onthe species and size of plants and the like, however, for example, whenArabidopsis thaliana is used as a host, paraquat may be present in amedium at a concentration of preferably 0.1 to 3.0 μM, more preferably1.0 to 3.0 μM, and most preferably 3.0 μM. A non-transgenic plant, i.e.,a wild-type plant, develops chlorosis and dies in a paraquat-containingmedium. In contrast, a plant transformed with the recombinant vectoraccording to the present invention remains green even in aparaquat-containing medium. Thus, it is possible to verify a cleardifference in growth in a paraquat-containing medium between anon-transgenic plant and a plant transformed with the recombinant vectoraccording to the present invention.

When employing antibiotic resistance or herbicide resistance as anindicator, false positivity may be observed at the screening stagebecause of the existence of a difference in sensitivity among the plant.In contrast, paraquat is a non-selective and potent herbicide that cankill all plants. Consequently, in the method of screening transgenicplants according to the present invention, false positivity is notobserved in the screening stage. Further, according to the method ofscreening transgenic plants according to the present invention,resistance can be effectively confirmed at an early stage of growth.

The promoter according to the present invention is a vascular tissue-and trichome-specific promoter comprising the DNA of the following (a),(b) or (c):

-   -   (a) DNA consisting of the nucleotide sequence represented by SEQ        ID NO: 3;    -   (b) DNA consisting of a nucleotide sequence having a        substitution, deletion or addition of one or a plurality of        nucleotides relative to the nucleotide sequence represented by        SEQ ID NO: 3 and functioning as a vascular tissue- and        trichome-specific promoter; and    -   (c) DNA hybridizing under stringent conditions to DNA consisting        of the nucleotide sequence represented by SEQ ID NO: 3 and        functioning as a vascular tissue- and trichome-specific        promoter.

The vascular tissue- and trichome-specific promoter described in theabove (a) is a vascular tissue- and trichome-specific promoter found inan untranslated region on the 5′-upstream side of the AtMVR gene andconsists of the nucleotide sequence represented by SEQ ID NO: 3. Thepromoter described in (a) can be determined by performing a search basedon approximately 3,000 nucleotides on the 5′-upstream side of the AtMVRgene on a database having the complete nucleotide sequence forArabidopsis thaliana.

As used herein, the term “vascular tissue- and trichome-specificpromoter” refers to a promoter exhibiting activity specific to avascular tissue and trichome of a plant. The term “vascular tissue”refers to a fascicular tissue system that differentiates through eachorgan of pteridophytes and spermatophytes, such as the stem, leaf, androot. Xylem and phloem are the components of the vascular tissue, andthey function as pipes to transport water and internal substancesthroughout the plant. Meanwhile, the term “trichome” refers to afloccose outgrowth existing on the surface of a leaf, stem, or sepal ofa plant body. A trichome participates in secretion and excretion fromthe plant body surface.

The activity of a vascular tissue- and trichome-specific promoter can bedetermined in accordance with a conventional method. For example, anexpression vector having a reporter gene operably linked theretodownstream of a promoter may be constructed. Next, an appropriate plantis transformed with the expression vector. The obtained transformant isthen cultured under predetermined conditions, and the expression amountof the reporter gene in a vascular tissue and trichome may be determinedat the mRNA or protein level to enable the measurement of promoteractivity under the relevant conditions. Further, when the reporter geneis the β-Glucuronidase (GUS) gene, the specificity of promoter activityin a vascular tissue and trichome can be determined by observing thehistochemical coloring caused by the expressed GUS.

For example, as a method of the histochemical coloring using GUS, amethod may be mentioned in which a reaction mixture containing5-brome-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc) as a GUS substrateis added to a tissue of a transformant in which the GUS gene has beenintroduced. When the GUS gene is expressed, X-Gluc is de-esterified togenerate an indoxyl derivative monomer, and this monomer isoxidation-polymerized with air to form a blue indigotin pigment. In atransformed cell or tissue, this blue pigment accumulates to exhibit ablue color.

Further, a specified untranslated region on the 5′-upstream side of theAtMVR gene can be readily obtained by conducting PCR employing genomeextracted from Arabidopsis thaliana as a template and using primers thatare complementary to the nucleotide sequences on both ends of theregion.

The promoter according to the present invention may be the nucleotidesequence represented by SEQ ID NO: 3, more specifically, the entireuntranslated region on the 5′-upstream side, or it may be one part ofDNA consisting of the nucleotide sequence represented by SEQ ID NO: 3 inso far as it exhibits a function as a vascular tissue- andtrichome-specific promoter.

The vascular tissue- and trichome-specific promoter described in theabove (b) consists of a nucleotide sequence having a substitution,deletion or addition of one or a plurality of nucleotides (for example,1 to 10, or 1 to 5) relative to the nucleotide sequence represented bySEQ ID NO: 3 and functions as a vascular tissue- and trichome-specificpromoter.

The vascular tissue- and trichome-specific promoter described in theabove (c) hybridizes under stringent conditions to DNA consisting of thenucleotide sequence represented by SEQ ID NO: 3 and functions as avascular tissue- and trichome-specific promoter.

As used herein, the term “stringent conditions” refers to, for example,when using probe DNA labeled with phosphorus-32, hybridization in ahybridization solution consisting of 5×SSC (0.75 M NaCl, 0.75 M sodiumcitrate), 5× Denhardt's reagent (0.1% Ficoll, 0.1% polyvinylpyrrolidone,0.1% bovine serum albumin) and 0.1% sodium dodecyl sulphate (SDS) at atemperature between 45 and 68° C., preferably 60 to 68° C. Further, inthe washing step, washing is performed in a washing solution consistingof 2×SSC and 0.1% SDS at a temperature between 45 and 55° C., and morepreferably in a washing solution consisting of 0.1×SSC and 0.1% SDS at atemperature between 45 and 55° C. When using probe DNA enzymaticallylabelled using the AlkPhos direct labeling module kit (AmershamBiotech), hybridization may be conducted in a hybridization solution(containing 0.5 M NaCl and 4% blocking reagent) having the compositiondescribed in the manual accompanying the kit at a temperature between 55to 75° C. Further, in a washing step, washing may be conducted in aprimary washing solution (containing 2 M urea) in accordance with theinstructions in the manual accompanying the kit at a temperature between55 to 75° C., and in a secondary washing solution at room temperature.Other detection techniques may also be used, in which case theconditions may be the standard conditions for the relevant detectiontechnique.

Once the nucleotide sequence of the promoter according to the presentinvention has been determined, it is then possible to obtain thepromoter according to the present invention by chemical synthesis, or byPCR employing a cloned probe as a template, or by performinghybridization employing a DNA fragment having the nucleotide sequence asa probe. Further, it is possible to synthesize a mutant of the promoteraccording to the present invention having equivalent functions as thoseprior to mutation by a technique such as site-directed mutagenesis.

Examples of the method for introducing a mutation into the promoteraccording to the present invention include a known method such as theKunkel method or the gapped duplex method or a method in accordance withsuch methods. For example, the introduction of a mutation can beperformed using a kit for introducing a mutation (for example, Mutant-Kor Mutant-G (both manufactured by TAKARA, Inc.) utilizing site-directedmutagenesis or using LA PCR in vitro Mutagenesis series kit manufacturedby TAKARA, Inc.

A recombinant vector according to the present invention comprising thepromoter according to the present invention can be obtained by insertingthe promoter according to the present invention into an appropriatevector. A vector for inserting the promoter according to the presentinvention is not particularly limited as long as it is capable ofreplication within a host, and examples thereof include a plasmid, ashuttle vector, and a helper plasmid. In addition, when the vectoritself is not capable of replication, a DNA fragment that is capable ofreplication by a method such as insertion into the chromosome of a hostmay be used.

Examples of plasmid DNA include a plasmid derived from E. coli (pBI221and the like, for example, pET system such as pET30b, pBR system such aspBR322 and pBR325, pUC system such as pUC118, pUC119, pUC18 and pUC19,pBluescript, and pBI221), a plasmid derived from Bacillus subtilis (forexample, pUB110 and pTP5), a binary plasmid derived from Agrobacteriumtumefaciens (for example, pBI system derived from pBIN19, pBI101, orpBI121), a plasmid derived from yeast (for example, YEp system such asYEp13, or YCp system such as YCp50) or the like. Examples of phage DNAinclude λ phage (Charon 4A, Charon 21A, EMBL3, EMBL4, λgt10, λgt11, λZAPand the like). Further, an animal virus vector such as retrovirus orvaccinia virus, or an insect virus vector such as baculovirus can alsobe used.

To insert the promoter according to the present invention into a vector,a method may be used in which purified DNA is first cleaved with anappropriate restriction enzyme and then inserted into a restrictionenzyme site or multicloning site of an appropriate vector DNA andligated to the vector. Further, a method may also be used in which ahomologous region is respectively provided in one part of a vector andthe promoter according to the present invention, and the vector andpromoter are ligated by an in vitro method using PCR or the like or anin vivo method using yeast or the like.

The recombinant vector according to the present invention comprising thepromoter according to the present invention can further include aforeign gene or a foreign DNA fragment that is inserted downstream ofthe promoter according to the present invention. A method for insertinga foreign gene or a foreign DNA fragment is the same as a method forinserting the promoter according to the present invention into a vector.

In the recombinant vector according to the present invention comprisingthe promoter according to the present invention, examples of a foreigngene to be inserted downstream of the promoter according to the presentinvention include any foreign gene, and specific examples include a geneinvolved in transport of water or internal substances or in cellproliferation, a gene involved in proliferation or transport of bacteriaor a plant virus, a gene involved in discharge of a heavy metal or thelike, or a gene involved in secretion of a liquid having antimicrobialactivity or a feeding deterrent effect. More specifically, the gene maybe a gene for transporter or pump, a gene encoding a PR-protein(Pathogenesis related protein) (chitinase, peroxidase or the like), adefensin family gene, a phytoalexin synthesis gene or a repellantpheromone synthesis gene of a pest insect or the like. As furtherexamples of a foreign gene, the gene according to the present inventiondescribed above, the AtMVR gene, may be mentioned.

Examples of the foreign DNA fragment to be inserted downstream of thepromoter according to the present invention include antisense RNA or aribozyme in which the RNA itself is functioning.

The transgenic plant according to the present invention is a transgenicplant having the recombinant vector according to the present inventioncomprising the promoter according to the present invention. Thetransgenic plant according to the present invention can be obtained byintroducing the recombinant vector according to the present inventioncomprising the promoter according to the present invention into a plant.A transgenic plant can be obtained in the manner described below.

A “plant” to be transformed in the present invention may be any of: awhole plant, a plant organ having a vascular tissue and/or trichome (forexample, leaf, petal, stem, root, or seed), plant tissue (for example,epidermis, phloem, parenchyma, or xylem) or a plant culture cell.Examples of the plant that can be used in the transformation include,but are not limited to, a plant belonging to the family Poaceae,Brassicaceae, Solanaceae, or Leguminosae (see below).

-   -   Poaceae: Oryza sativa, Zea mays    -   Brassicaceae: Arabidopsis thaliana    -   Solanaceae: Nicotiana tabacum    -   Leguminosae: Glycine max

The recombinant vector according to the present invention comprising thepromoter according to the present invention can be introduced into aplant by a conventional transformation method such as, for example, theelectroporation method, Agrobacterium method, particle gun method, orPEG method.

For example, when using the electroporation method the recombinantvector according to the present invention comprising the promoteraccording to the present invention is introduced into a host by thetreatment using an electroporation apparatus equipped with a pulsecontroller under conditions of a voltage of 500 to 1600 V, at 25 to 1000μF, for 20 to 30 msec.

When using the particle gun method, the whole plant, a plant organ orplant tissue itself may be used without any treatment, a section thereofmay be prepared and then used, or protoplast may be prepared and used.The prepared sample can then be treated using a gene transfer device(for example, PDS-1000/He manufactured by Bio-Rad Inc.). Although thetreatment conditions may vary depending on the plant or sample used, thetreatment is normally conducted at a pressure of approximately 450 to2000 psi and a distance of approximately 3 to 12 cm.

The method using the Ti plasmid or Ri plasmid of Agrobacterium takesadvantage of a characteristic whereby, when a bacterium belonging to thegenus Agrobacterium infects a plant, one part of plasmid DNA possessedby the bacterium is transferred into the genome of the plant. Thismethod can thus be used to introduce the promoter according to thepresent invention and a foreign gene or foreign DNA fragment into aplant host. Among the bacteria belonging to the genus Agrobacterium,when Agrobacterium tumefaciens infects a plant, it causes the formationof a tumor that is referred to as “crown gall.” Further, whenAgrobacterium rhizogenes infects a plant, it incites the generation of acapillary root. These are caused by a region referred to as a “T-DNA(Transferred DNA) region” of a Ti plasmid or Ri plasmid transferringinto a plant at the time of infection to be integrated into the genomeof the plant. Accordingly, the DNA to be integrated into a plant genomeis first inserted into the T-DNA region of a Ti plasmid or Ri plasmid,and then the DNA can be integrated into the plant genome by infectingthe plant host with a bacterium of the genus Agrobacterium.

Examples of the method for transforming a bacterium of the genusAgrobacterium into a plant host include the above describedelectroporation method, particle gun method and PEG method, as well asan in planta method. Examples of the in planta method include a directAgrobacterium inoculation method and an infiltration method.

The tumor tissue or shoot, capillary root or the like obtained as theresult of transformation can be used without any treatment for cellculture, tissue culture or organ culture. Alternatively, it can beregenerated in a plant body by administration of a plant hormone (auxin,cytokinin, gibberellin, abscisic acid, ethylene, brassinolide or thelike) of an appropriate concentration using a plant tissue culturemethod known in the prior art.

Further, the promoter according to the present invention and a foreigngene or foreign DNA fragment can be introduced into a plant by utilizinga plant virus as a vector. Examples of the plant virus that can be usedherein include cauliflower mosaic virus. First, the viral genome isinserted into a vector derived from E. coli or the like to produce arecombinant, and then the promoter according to the present inventionand the foreign gene or foreign DNA fragment is inserted into the viralgenome. The viral genome modified in this manner is subsequently cleavedfrom the recombinant using a restriction enzyme, and the promoteraccording to the present invention and the foreign gene or foreign DNAfragment can be introduced into a plant host by inoculating the viralgenome into the plant host.

The transgenic plant according to the present invention produced in theabove manner can specifically express the foreign gene or foreign DNAfragment in a vascular tissue and trichome using the promoter accordingto the present invention.

The vascular tissue is a location involved in transporting water andinternal substances as well as cell proliferation in a plant. Thus, whena gene involved in transporting water or internal substances or in cellproliferation is introduced as a foreign gene into the transgenic plantaccording to the present invention, the transport of water and internalsubstances or cell proliferation in the plant can be regulated. Thevascular tissue is also a site where wilt disease fungus infectingplants of the family Solanaceae proliferates and transfers. When a plantvirus infects a plant, the plant virus migrates a long distance from oneleaf to an above leaf and therefore the vascular tissue is also amigration site that leads to systemic infection of a plant. Thus, when agene involved in proliferation or migration of a fungus or plant virusis introduced as a foreign gene into the transgenic plant according tothe present invention, the plant can be protected from the fungus orplant virus.

Meanwhile, a trichome is involved in secretion and excretion from thesurface of a plant body. For example, it is reported that when a plantis exposed to heavy metal (cadmium) stress, the number of trichomes onthe surface of leaves increases and crystals containing cadmium orcalcium adhere to the surface of the leaves, in other words, thatcadmium is excreted by a trichome (Planta, 213 (1), 45-50, 2001, May). Atrichome is also the site of first contact for a filamentous fungus,bacterium, insect or the like invading a plant. Further, as a defenseagainst diseases and insect damages, for example, a fluid havingantimicrobial activity and a feeding deterrent effect is secreted from aglandular hair or glandular trichome of rugosa rose of the familyRosaceae, one type of trichome. Therefore, when a gene involved in theexcretion of a heavy metal or the like, or a gene involved in secretionof a fluid having antimicrobial activity or a feeding deterrent effectis introduced as a foreign gene into the transgenic plant according tothe present invention, heavy metal can be efficiently excreted from theplant or the plant can be effectively protected against a filamentousfungus, bacterium or insect invading the plant.

Further, when the gene according to the present invention is introducedas a foreign gene into the transgenic plant according to the presentinvention, resistance to paraquat can be imparted by promoting thetransportation and excretion and the like of paraquat incorporated inthe plant body.

EXAMPLES

The present invention will be explained in detail further below withreference to the following examples. However, the examples are notintended to limit the technical scope of the invention.

Example 1 Isolation of Paraquat Resistance Gene

In this example, Weigel T-DNA lines acquired from Nottingham ArabidopsisStock Center (http://nasc.nott.ac.uk/) were used as activation tag linesof Arabidopsis thaliana.

(1) Screening of Individuals Capable of Growing in Paraquat-ContainingMedium Using Activation-Tagged Lines of Arabidopsis thaliana (WeigelT-DNA Lines)

Seeds of Weigel T-DNA lines were sterilely inoculated in ½ MS agar (1%)culture medium (2.3 g/l of Murashige and Skoog Plant Salt Mixture(manufactured by Wako Pure Chemical Industries Ltd.), 1.5 mg/l ofthiamine hydrochloride, 2.5 mg/l of nicotinic acid, 0.25 mg/l ofpyridoxine hydrochloride, 1.5% of sucrose, 1% of agar) containing 3 μMof paraquat (methyl viologen, manufactured by Sigma Chemical Co.), andcultured at 22° C. under irradiation of light of 60 μE/m²/s (cycle of 16hrs photoperiod/8 hrs dark period). Approximately 10 days after culture,individuals growing in the paraquat-containing medium were screened.

(2) Estimation of Insertion Sites of T-DNA from the ScreenedActivation-Tagged Lines by the TAIL-PCR Method

Seeds of Weigel T-DNA lines from which screened individuals originatedwere planted in a pot containing vermiculite (manufactured by AsahiKagaku Kogyo Co., Ltd.) and grown for approximately one month at 23° C.under a light intensity of 100 μE/m²/s with a photoperiod condition of16 hrs photoperiod/8 hrs dark period.

Genome DNA was prepared from leaves of cultivated individuals using theDNeasy Plant Mini Kit (manufactured by QIAGEN), and three types ofspecific primers (TL1: SEQ ID NO: 31; TL2: SEQ ID NO: 32; TL3: SEQ IDNO: 33) were designed for the vicinity of a T-DNA left sequence (T-DNAleft border: SEQ ID NO: 30) of an activation-tagging vector (pSKI015:GenBank accession No. AF187951) used with the Weigel T-DNA lines.TAIL-PCR (Shokubutsu No PCR Jikken Purotokoru (Protocols of PCRExperiments for Plants), (Eds. Shimamoto K. & Sasaki T.), New Edition,2000, pp 83-89, Shujunsha Co., Ltd., Tokyo; Genomics, 25, 674-681, 1995;Plant J., 8, 457-463, 1995) was then performed using the specificprimers and a random primer 1 (SEQ ID NO: 34) and the PCR reactionmixture and reaction conditions described below to amplify genome DNAbordering the T-DNA. In SEQ ID NO: 34, n represents a, g, c, or t(location: 1 and 11), s represents g or c (location: 7), and wrepresents a or t (location: 8 and 13).

The composition of the reaction mixture and the PCR conditions for thefirst-round PCR are listed in tables 2 and 3. TABLE 2 Template (genomeDNA): 10 ng 10x PCR buffer (manufactured by TAKARA BIO Inc.): 2 μl 2.5mM dNTPs (manufactured by TAKARA BIO Inc.): 1.6 μl First specific primer(TL1: SEQ ID NO: 31): 3 pmol Random primer 1 (SEQ ID NO: 34): 80 pmolAmpliTaq (manufactured by Applied Biosystems): 0.8 units Total volume 20μl

TABLE 3 #1: 94° C. (1 min)/95° C. (1 min) #2: 94° C. (1 min)/65° C. (1min)/72° C. (3 min) × 5 cycles #3: 94° C. (1 min)/25° C. (3 min) → to72° C. at 3 min/72° C. (3 min) × 1 cycle #4: 94° C. (30 sec)/68° C. (1min)/72° C. (3 min) 94° C. (30 sec)/68° C. (1 min)/72° C. (3 min) 94° C.(30 sec)/44° C. (1 min)/72° C. (3 min) × 14 cycles #5 72° C. (5 min)

The composition of the reaction mixture and the PCR conditions for thesecond-round PCR are listed in tables 4 and 5. TABLE 4 Template (a50-fold dilution of product of first-round PCR): 1 μl 10x PCR buffer: 2μl 250 μM dNTPs: 2 μl Second specific primer (TL2: SEQ ID NO: 32): 4pmol Random primer 1 (SEQ ID NO: 34): 60 pmol AmpliTaq: 0.6 units Totalvolume 20 μl

TABLE 5 #6: 94° C. (30 sec)/64° C. (1 min)/72° C. (3 min) 94° C. (30sec)/64° C. (1 min)/72° C. (3 min) 94° C. (30 sec)/44° C. (1 min)/72° C.(3 min) × 10 cycles #5 72° C. (5 min)

The composition of the reaction mixture and the PCR conditions for thethird-round PCR are listed in tables 6 and 7. TABLE 6 Template (a50-fold dilution of product of 1 μl second-round PCR): 10x PCR buffer:10 μl 2.5 mM dNTPs: 1 μl Third specific primer (TL3: SEQ ID NO: 33): 30pmol Random primer 1 (SEQ ID NO: 34): 300 pmol AmpliTaq: 3 units Totalvolume 100 μl

TABLE 7 #7: 94° C. (1 min)/44° C. (1 min)/72° C. (3 min) × 20 cycles #572° C. (5 min)

Next, after subjecting the reaction products from the second-round PCRand third-round PCR to electrophoresis on agarose gel, the presence orabsence of amplification and the specificity of the reaction productswere verified.

Further, using the specific primer TL3 (SEQ ID NO: 33), theamplification product of the third-round PCR was directly sequencedusing the ABI PRISM Dye Terminator Cycle Sequencing Kit (AppliedBiosystems) and the nucleotide sequence was then determined using theABI PRISM 310 genetic analyzer (Applied Biosystems). As a result, 278-bpsequence information was obtained (SEQ ID NO: 35). In SEQ ID NO: 35, nrepresents a, g, c, or t (location: 13, 35, 73, 108, 156, 190, 198 and201). A search was performed for the obtained sequence on databaseshaving the entire nucleotide sequence of Arabidopsis thaliana, and itwas found that the insertion site is located at 77240 bp of BAC cloneF17123.

(3) Isolation of cDNA of Paraquat Resistance Gene

Seeds of Arabidopsis thaliana (Arabidopsis thaliana ecotype Columbia(Col-0)) were planted in a pot containing vermiculite (Asahi KagakuKogyo Co., Ltd.) and allowed to grow for approximately one month at 23°C. under a light intensity of 100 μE/m²/s with a photoperiod conditionof 16 hrs photoperiod/8 hrs dark period.

After growing, leaves of individuals were frozen using liquid nitrogen.Subsequently, total RNA was extracted using the RNeasy Plant Mini Kit(manufactured by QIAGEN). Thereafter, cDNA was synthesized from theextracted total RNA using the ProSTAR First Strand RT-PCR Kit(manufactured by STRATAGEN).

Based on the sequence of a putative open reading frame (ORF) genepresent within an adjacent 10 kb of a structural gene having thenucleotide sequence (SEQ ID NO: 35) obtained in the above (2), a primer141dF (SEQ ID NO: 36) and a primer 141dR (SEQ ID NO: 37) were designedfor the putative structural gene, and PCR was then performed using theseprimers and the following reaction mixture (Table 8) containing TakaraEX-Taq (manufactured by TAKARA BIO Inc.) employing the above synthesizedcDNA as a template. TABLE 8 Template (cDNA): 50 ng 10x Ex Taq buffer(TAKARA BIO Inc.): 2 μl dNTPs: 200 μM Each primer: 0.2 μM Takara EX-Taq:1 unit Total volume 20 μl

Thirty cycles of 94° C. (30 sec)/55° C. (30 sec)/72° C. (60 sec) wereemployed as the reaction conditions.

The amplification product was cloned into the pGEM-T Easy vector(manufactured by Promega), and the nucleotide sequence was thendetermined using the ABI PRISM 310 genetic analyzer (AppliedBiosystems). As a result, a cDNA fragment of 857 bp was obtained (SEQ IDNO: 1). This cDNA fragment was designated as AtMVR gene, and the pGEM-TEasy vector containing AtMVR gene was designated as pAtMVR. The aminoacid sequence encoded by the AtMVR gene is shown in SEQ ID NO: 2.

Example 2 Search for AtMVR Homologous Gene with Respect to AtMVR Gene

The search was made on databases having the entire nucleotide sequenceof Arabidopsis thaliana based on the nucleotide sequence of the AtMVRgene and found that in addition to the nucleotide sequence of the AtMVRgene there are 13 AtMVR homologous genes on the Arabidopsis thalianagenome.

The respective AtMVR homologous genes were designated as AtMVR3-1 toAtMVR3-13. The nucleotide sequence of each of the AtMVR homologous genesand the putative amino acid sequence encoded by the relevant AtMVRhomologous gene are shown by the SEQ ID NOs. listed in Table 9 below.Table 9 also lists the results of homology analysis between the AtMVRgene and each AtMVR homologous gene. The homology analysis was conductedusing BLAST P at the amino acid level. The term “Identities” refers to100% correspondence in terms of amino acids. Amino acids may beclassified based on the chemical properties of their side chains. In theBLOSUM62 amino acid substitution matrix, amino acids are classifiedinto: an amino acid with a mercapto group (C); hydrophilic amino acidsthat have low molecular weights (S, T, P, A, G); acidic amino acids (N,D, E, Q); basic amino acids (H, R, K); hydrophobic amino acids that havelow molecular weights (M, I, L, V); and aromatic amino acids (F, Y, W).In Table 9, the term “Identities” refers to 100% correspondence in termsof amino acids and the term “Positives” refers to the numerical valuewhen amino acids having a positive score in the BLOSUM 62 amino acidsubstitution matrix are added to those having 100% correspondence. TABLE9 Name of AtMVR homologous gene Nucleotide sequence Amino acid sequenceIdentities (%) Positives (%) AtMVR3-1 SEQ ID NO: 4 SEQ ID NO: 5 57 70AtMVR3-2 SEQ ID NO: 6 SEQ ID NO: 7 55 69 AtMVR3-3 SEQ ID NO: 8 SEQ IDNO: 9 39 56 AtMVR3-4 SEQ ID NO: 10 SEQ ID NO: 11 39 55 AtMVR3-5 SEQ IDNO: 12 SEQ ID NO: 13 37 54 AtMVR3-6 SEQ ID NO: 14 SEQ ID NO: 15 36 52AtMVR3-7 SEQ ID NO: 16 SEQ ID NO: 17 34 52 AtMVR3-8 SEQ ID NO: 18 SEQ IDNO: 19 34 51 AtMVR3-9 SEQ ID NO: 20 SEQ ID NO: 21 37 58 AtMVR3-10 SEQ IDNO: 22 SEQ ID NO: 23 25 44 AtMVR3-11 SEQ ID NO: 24 SEQ ID NO: 25  33* 51* AtMVR3-12 SEQ ID NO: 26 SEQ ID NO: 27 25 41 AtMVR3-13 SEQ ID NO: 28SEQ ID NO: 29 22 41*The comparison with AtMVR3-11 shows the result of homology comparisonwith a partial sequence of AtMVR3-11.

As shown in Table 9, homology between AtMVR and the 13 AtMVR homologousgenes ranged from 22 to 57% for Identities and from 41 to 70% forPositives.

Example 3 Construction of AtMVR Expression Vector for Plant andProduction of AtMVR Transgenic Plant

The transformation techniques applied herein were in accordance with avector system described by Pellegrineschi et al. (Biochemical SocietyTransitions 23, 247-250, 1995) based on the Agrobacterium gene transportsystem outlined by Hinchee et al. (Plant Cell and Tissue Culture, pp.231-270, Eds. I. K. Vasil, T. A Thorpe, Kluwer Academic Publisher,1994).

(1) Construction of AtMVR Expression Vector for Plant

The AtMVR gene sequence was excised from pAtMVR using SacI/SacII andsubcloned into pBlueScript (STRATAGENE). Subsequently, a fragmentcontaining the AtMVR gene sequence was cleaved with XbaI/SacI andintroduced at XbaI/SacI site that is present downstream of the CaMV 35Spromoter of pBI121 (manufactured by Clontech). The resulting vector wasused below as an AtMVR expression vector for plant.

(2) Production of AtMVR Transgenic Plant

The AtMVR expression vector for plant produced in the above (1) wasintroduced into Agrobacterium tumefaciens LBA4404 strain by theelectroporation method (Plant Molecular Biology Manual, Second Edition,B. G. Stanton, A. S. Robbert, Kluwer Academic Publishers, 1994).Subsequently, the Agrobacterium tumefaciens having the AtMVR expressionvector for plant introduced therein was introduced into wild-typeArabidopsis thaliana ecotype Col-0 by an infiltration method describedby Clough et al. (The Plant Journal 16: 735-743, 1998).

Transformants were screened in a kanamycin-containing medium, and a T3generation plant (homozygous line having 1 AtMVR gene introduced) wasproduced by self-pollination.

Next, the amount of the introduced AtMVR gene expressed was examined.Seeds of a transformant produced as described above and anon-transformant were respectively planted in pots containingvermiculite (Asahi Kagaku Kogyo Co., Ltd.) and allowed to grow forapproximately one month under a light intensity of 100 μE/m²/s at 23° C.with a photoperiod condition of 16 hrs photoperiod/8 hrs dark period.

After growing, total RNA was extracted from the transformant and thewild-type Arabidopsis thaliana ecotype Col-0 non-transformant using theRNeasy Plant Mini Kit (QIAGEN). Thereafter, 1 μg of RNA was subjected toreverse transcription using the ProSTAR First Strand RT-PCR Kit(STRATAGEN). PCR was then performed using the following reaction mixture(Table 10) containing Takara EX-Taq (TAKARA BIO) employing {fraction(1/50)} volume of the synthesized cDNA as a template and using primersfor the AtMVR gene (primer 141d1 (SEQ ID NO: 38) and primer 141d2 (SEQID NO: 39)). TABLE 10 PCR reaction mixture: Template (cDNA): 50 ng 10xEx Taq buffer (TAKARA BIO Inc.): 2 μl dNTPs: 200 μM Each primer: 0.2 μMTakara EX-Taq: 1 unit Total volume 20 μl

Thirty cycles of 94° C. (30 sec)/55° C. (30 sec)/72° C. (60 sec) wereemployed as the reaction conditions.

The amplification products were subjected to electrophoresis on agarosegel. FIG. 1 shows the results of electrophoresis. As can be seen fromFIG. 1, in comparison to the non-transformant, the produced transformantoverexpressed the AtMVR gene.

Example 4 Evaluation of Paraquat Resistance of AtMVR Gene Transformant

Seeds derived from the produced AtMVR gene transformant andnon-transformant (wild-type Arabidopsis thaliana ecotype Col-0) weresterilely inoculated in ½ MS agar (1%) medium (2.3 μl of Murashige andSkoog Plant Salt Mixture (Wako Pure Chemical Industries Ltd.), 1.5 mg/lof thiamine hydrochloride, 2.5 mg/l of nicotinic acid, 0.25 mg/l ofpyridoxine hydrochloride, 1.5% of sucrose) containing 3 μM of paraquat(methyl viologen (Sigma Chemical Co.)), and cultured for eight days at22° C. under irradiation of light of 60 μmol/m²/s (cycle of 16 hrsphotoperiod/8 hrs dark period). After culture, the growth of germinatedindividuals was evaluated. The results are shown in FIG. 2, wherein FIG.2B is a photograph showing growth of an AtMVR gene transformant and anon-transformant in a ½ MS culture medium without paraquat, and FIG. 2Cis a photograph showing growth of an AtMVR gene transformant and anon-transformant in a ½ MS culture medium with paraquat. FIG. 2A is aschematic diagram showing the location of the AtMVR gene transformantand the non-transformant in FIGS. 2B and 2C.

As can be seen from FIG. 2B, the results showed that in the culturemedium without paraquat, the AtMVR gene transformant exhibited the samegrowth as the non-transformant. Meanwhile, as can be seen from FIG. 2C,in a medium containing paraquat the non-transformant developed chlorosisand died, i.e. growth was remarkably inhibited, while in contrast theseedling of the AtMVR gene transformant was able to grow. Thus, it wasconfirmed that in a medium without paraquat, the AtMVR gene transformantexhibited the same growth as a non-transformant regardless of expressionof the AtMVR gene, and also that in a medium with paraquat, the AtMVRgene transformant had clearly greater paraquat resistance than thenon-transformant.

Example 5 Isolation of Vascular Tissue/Trichome-Specific Promoter

Seeds of Arabidopsis thaliana (Arabidopsis thaliana ecotype Columbia(Col-0)) were planted in pots containing vermiculite (Asahi Kagaku KogyoCo., Ltd.) and allowed to grow for approximately one month under a lightintensity of 100 μE/m²/s at 23° C., under a photoperiod condition of 16hrs photoperiod/8 hrs dark period.

After growing, genome DNA was prepared from leaves of individuals usingDNeasy Plant Mini Kit (QIAGEN). Next, PCR was performed using thefollowing reaction mixture (Table 11) containing Takara EX-Taq (TAKARABIO Inc.) employing the obtained genome DNA as a template and using aprimer 141dpF (SEQ ID NO: 40) and a primer 141dpR (SEQ ID NO: 41) basedon the AtMVR gene fragment (SEQ ID NO: 35) described in above Example 1under the reaction conditions described below. TABLE 11 Template genomeDNA: 50 ng 10x Ex Taq buffer (TAKARA BIO Inc.): 2 μl dNTPs: 200 μM Eachprimer: 0.2 μM Takara EX-Taq: 1 unit Total volume 20 μl

The reaction conditions were thirty cycles of 94° C. (30 sec)/55° C. (30sec)/72° C. (60 sec).

The amplification product was cloned into pGEM-T Easy vector (PromegaCorp.) and the nucleotide sequence was then determined using the ABIPRISM 310 genetic analyzer (Applied Biosystems). As a result, an AtMVRpromoter of 1722 bp (SEQ ID NO: 3) was obtained.

Example 6 Analysis of Tissue Specificity of VascularTissue/Trichome-Specific Promoter

(1) Construction of Expression Vector Having AtMVR Promoter

The AtMVR promoter was excised from the pGEM-T Easy vector having theAtMVR promoter (SEQ ID NO: 3) produced in Example 5 using HindIII/PstIand then subcloned into the upstream region of CaMV 35S promoter ofpBI221 (Clontech). The resulting vector was treated with PstI/SmaI toremove the CaMV 35S promoter, and the ends were blunted using DNA T4polymerase (TAKARA BIO Inc.) and allowed to self-ligate. As a result,the AtMVR promoter was ligated into the vector upstream of theβ-Glucuronidase (GUS) gene. Subsequently, a fragment containing theAtMVR promoter and the GUS gene was excised from the above vector usingHindIII/EcoRI, and then substituted for CaMV 35S promoter-β-GUS gene ofpBI121 (Clontech). The vector thus obtained was employed as anexpression vector having the AtMVR promoter for use below.

(2) Production of Transgenic Plant

In a similar manner to Example 3(2), the above expression vector havingthe AtMVR promoter was introduced into Agrobacterium tumefaciens LBA4404strain, and this was then introduced into wild-type Arabidopsisthaliaiia ecotype Col-0 by the infiltration method. Thereafter,transformants were screened in a kanamycin-containing medium, and a T3generation plant was produced by self-pollination.

(3) Analysis of Tissue Specificity of AtMVR Promoter

Seeds derived from the transformant produced in the above (2) weresterilely inoculated in ½ MS agar (1%) medium (2.3 g/l of Murashige andSkoog Plant Salt Mixture (Wako Pure Chemical Industries Ltd.), 1.5 mg/lof thiamine hydrochloride, 2.5 mg/l of nicotinic acid, 0.25 mg/l ofpyridoxine hydrochloride, 1.5% of sucrose), and cultured at 22° C. underirradiation of light of 60 μE/m²/s (cycle of 16 hrs photoperiod/8 hrsdark period) for approximately 7 days.

After culture, transformants that grew were fixed with acetone. Areaction mixture containing 5-brome-4-chloro-3-indolyl-β-D-glucuronide(X-Gluc) was added to the fixed tissue so as to immerse the entiretissue. The composition of the reaction mixture was: 1.9 mM of X-Gluc,0.5 mM of K₃Fe(CN)₆, 0.5 mM of K₄Fe(CN)₆, and 0.3% of Triton X-100.

The container was then sealed and incubated overnight in a 37° C.incubator. Thereafter, 70% ethanol was added to the mixture to terminatethe reaction, and coloring was observed. (Shokubutsu No Saibo Wo MiruJikken Purotokoru (Protocols of Experiments for Observing Cells ofPlants), Eds. Fukuda H., Nishimura M., & Nakamura K., (1997), pp 71-79,Shujunsha Co., Ltd., Tokyo). The results are shown in FIGS. 3 to 5,wherein FIGS. 3 to 5 are photomicrographs of an entire transformant, aleaf of a transformant, and a root of a transformant, respectively.

As can be seen from FIGS. 3 to 5, specific coloring was observed in thevascular tissue and trichome. Thus, it was confirmed that the obtainedAtMVR promoter (SEQ ID NO: 3) is a transcriptional promoter havingtissue-specific transcriptional activity in a vascular tissue andtrichome.

Free Text for Sequence Listing

SEQ ID NOS: 31 to 41 are primers.

In SEQ ID NO: 34, n represents a, g, c, or t (location: 1 and 11), srepresents g or c (location: 7), and w represents a or t (location: 8and 13).

In SEQ ID NO: 35, n represents a, g, c, or t (location: 13, 35, 73, 108,156, 190, 198 and 201).

Industrial Applicability

According to the present invention there is provided a paraquatresistance gene and a vascular tissue- and trichome-specific promoter. Aparaquat resistance gene according to the present invention is capableof imparting resistance that is specific to paraquat without affectinggrowth regulation that undergoes control by the generation of activeoxygens under various environments.

Further, the vascular tissue- and trichome-specific promoter accordingto the present invention enables the regulation of gene expression in avascular tissue and trichome of a plant.

1. A gene encoding a protein of the following (a) or (b): (a) a proteinconsisting of the amino acid sequence represented by SEQ ID NO: 2, and(b) a protein consisting of an amino acid sequence having asubstitution, deletion or addition of one or a plurality of amino acidsrelative to the amino acid sequence represented by SEQ ID NO: 2 andimparting paraquat resistance.
 2. A protein imparting paraquatresistance encoded by the gene as recited in claim
 1. 3. A recombinantvector comprising the gene as recited in claim
 1. 4. The recombinantvector according to claim 3, wherein the recombinant vector furthercomprises a foreign gene or a foreign DNA fragment.
 5. A transformanthaving the recombinant vector as recited in claim 3 or
 4. 6. A plantbody having the recombinant vector as recited in claim 3 or 4 and havingparaquat resistance.
 7. A method for screening for a transgenic plant,comprising introducing the recombinant vector as recited in claim 4 intoa plant and screening for a transgenic plant on the basis of paraquatresistance as an indicator.
 8. A vascular tissue- and trichome-specificpromoter comprising DNA of the following (a), (b) or (c): (a) DNAconsisting of the nucleotide sequence represented by SEQ ID NO: 3; (b)DNA consisting of a nucleotide sequence having a substitution, deletionor addition of one or a plurality of nucleotides relative to thenucleotide sequence represented by SEQ ID NO: 3 and functioning as avascular tissue- and trichome-specific promoter, and (c) DNA hybridizingunder stringent conditions to DNA consisting of the nucleotide sequencerepresented by SEQ ID NO: 3 and functioning as a vascular tissue- andtrichome-specific promoter.
 9. A recombinant vector comprising thevascular tissue- and trichome-specific promoter as recited in claim 8.10. The recombinant vector according to claim 9, wherein the recombinantvector comprises a foreign gene or a foreign DNA fragment downstream ofthe vascular tissue- and trichome-specific promoter.
 11. The recombinantvector according to claim 10, wherein the foreign gene is the gene asrecited in claim
 1. 12. A transgenic plant having the recombinant vectoras recited in any one of claims 9 to 11.